克氏原螯虾(Procambarus clarkii)应急二价铜离子时超氧化物歧化酶和过氧化氢酶的变化

用二价铜离子(Cu2+)胁迫克氏原螯虾,分析其肝胰腺、触角腺、鳃等组织的超氧化物歧化酶(superoxide dismutase,SOD)和过氧化氢酶(catalase,CAT)活性变化.结果表明:克氏原螯虾能在含10mg/LCu2+水中生活;在10mg/LCu2+胁迫下,在0～24h,肝胰腺和触角腺中的CAT、SOD活性均显著升高,CAT活性在24h达到最高;在24～36h,肝胰腺和触角腺中的CAT活性开始下降,而SOD活性继续上升,在第36h时达到最高;48h后,肝胰腺和触角腺中SOD和SOD活性下降并均趋于平稳表达,SOD活性仍显著高于非胁迫处理时,而CAT活性则显著低于Cu2+处理时;鳃中的SOD和SOD活性未受到Cu2+浓度变化的影响.     

Theoretical mechanisms of the superoxide radical anion catalyzed by the copper‐zinc superoxide dismutase

The disproportionation of superoxide radical anions catalyzed by copper‐zinc superoxide dismutase was investigated in detail using density functional theory. The structures of each stationary point and the transition states were located so that the reaction pathways were determined. The results indicate that the reactions proceed by two steps both for the oxidized process of superoxide radical anion and the reduced one. The rate for the determining step of the reaction (2) is in very good agreement with the experimental value. The solvation effect on the reaction was also discussed. © 2009 Wiley Periodicals, Inc. Int J Quantum Chem, 2010     

Direct Detection of the Superoxide Anion as a Stable Intermediate in the Electroreduction of Oxygen in a Non‐Aqueous Electrolyte Containing Phenol as a Proton Source

The non‐aqueous Li–air (O₂) battery has attracted intensive interest because it can potentially store far more energy than today′s batteries. Presently Li–O₂ batteries suffer from parasitic reactions owing to impurities, found in almost all non‐aqueous electrolytes. Impurities include residual protons and protic compounds that can react with oxygen species, such as the superoxide (O₂^?), a reactive, one‐electron reduction product of oxygen. To avoid the parasitic reactions, it is crucial to have a fundamental understanding of the conditions under which reactive oxygen species are generated in non‐aqueous electrolytes. Herein we report an in situ spectroscopic study of oxygen reduction on gold in a dimethyl sulfoxide electrolyte containing phenol as a proton source. It is shown directly that O₂^?, not HO₂, is the first stable intermediate during the oxygen reduction process to hydrogen peroxide. The unusual stability of O₂^? is explained using density functional theory (DFT) calculations.     

Parsing disease‐relevant protein modifications from epiphenomena: perspective on the structural basis of SOD1‐mediated ALS

Conformational change and modification of proteins are involved in many cellular functions. However, they can also have adverse effects that are implicated in numerous diseases. How structural change promotes disease is generally not well‐understood. This perspective illustrates how mass spectrometry (MS), followed by toxicological and epidemiological validation, can discover disease‐relevant structural changes and therapeutic strategies. We (with our collaborators) set out to characterize the structural and toxic consequences of disease‐associated mutations and post‐translational modifications (PTMs) of the cytosolic antioxidant protein Cu/Zn‐superoxide dismutase (SOD1). Previous genetic studies discovered >180 different mutations in the SOD1 gene that caused familial (inherited) amyotrophic lateral sclerosis (fALS). Using hydrogen–deuterium exchange with mass spectrometry, we determined that diverse disease‐associated SOD1 mutations cause a common structural defect – perturbation of the SOD1 electrostatic loop. X‐ray crystallographic studies had demonstrated that this leads to protein aggregation through a specific interaction between the electrostatic loop and an exposed beta‐barrel edge strand. Using epidemiology methods, we then determined that decreased SOD1 stability and increased protein aggregation are powerful risk factors for fALS progression, with a combined hazard ratio > 300 (for comparison, a lifetime of smoking is associated with a hazard ratio of ~15 for lung cancer). The resulting structural model of fALS etiology supported the hypothesis that some sporadic ALS (sALS, ~80% of ALS is not associated with a gene defect) could be caused by post‐translational protein modification of wild‐type SOD1. We developed immunocapture antibodies and high sensitivity top‐down MS methods and characterized PTMs of wild‐type SOD1 using human tissue samples. Using global hydrogen–deuterium exchange, X‐ray crystallography and neurotoxicology, we then characterized toxic and protective subsets of SOD1 PTMs. To cap this perspective, we present proof‐of‐concept that post‐translational modification can cause disease. We show that numerous mutations (N➔D; Q➔E), which result in the same chemical structure as the PTM deamidation, cause multiple diseases. Copyright © 2017 John Wiley & Sons, Ltd.     

Absolute quantification of superoxide dismutase in cytosol and mitochondria of mice hepatic cells exposed to mercury by a novel metallomic approach

In the last years, the development of new methods for analyzing accurate and precise individual metalloproteins is of increasing importance, since numerous metalloproteins are excellent biomarkers of oxidative stress and diseases. In that way, methods based on the use of post column isotopic dilution analysis (IDA) or enriched protein standards are required to obtain a sufficient degree of accuracy, precision and high limits of detection. This paper reports the identification and absolute quantification of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) in cytosol and mitochondria from mice hepatic cells using a innovative column switching analytical approach. The method consisted of orthogonal chromatographic systems coupled to inductively coupling plasma-mass spectrometry equipped with a octopole reaction systems (ICP-ORS-MS) and UV detectors: size exclusion fractionation (SEC) of the cytosolic and mitochondrial extracts followed by online anion exchange chromatographic (AEC) separation of Cu/Zn containing species. After purification, Cu,Zn-SOD was identified after tryptic digestion by molecular mass spectrometry (MS). The MS/MS spectrum of a doubly charged peptide was used to obtain the sequence of the protein using the MASCOT searching engine. This optimized methodology reduces the time of analysis and avoids the use of sample preconcentration and clean-up procedures, such as cut-off centrifuged filters, solid phase extraction (SPE), precipitation procedures, off-line fractions insolates, etc. In this sense, the method is robust, reliable and fast with typical chromatographic run time less than 20 min. Precision in terms of relative standard deviation (n = 5) is of 3–5% and detection limits is 0.21 ng Cu g⁻¹.     

Detection of synergistic effect of superoxide dismutase and jujubosides on scavenging superoxide anion radical by capillary electrophoresis

A new capillary electrophoresis method was developed to study the synergistic effect of superoxide dismutase and jujuboside A or B on scavenging superoxide anion radical in serum matrix respectively, in which superoxide anion radical was generated from pyrogallol autoxidation. The electrophoresis conditions, and the factors affecting the productive rate of purpurogallin, such as pyrogallol autoxidation product and the activity of superoxide dismutase, were optimized. Under optimal conditions, the content of superoxide dismutase in Gibco newborn calf serum was 7.06 mg/L, RSD was 2.01% and the average recovery was 98.4%. The values of IC₅₀ for jujuboside A and B in the serum matrix were 157.67 and 31.60 mg/L respectively, and they both had synergy on scavenging superoxide anion radical with superoxide dismutase, but there was no the dose‐dependency on this synergy.     

甲烷氧化菌素功能化纳米金修饰电极模拟SOD的电化学性能研究

利用混合自组装的方式,将Mb功能化纳米金（Mb-AuNPs-MUA）修饰在金电极表面,以制备出检测超氧阴离子无电子媒介体生物传感器.采用UV-Vis考察修饰纳米团簇的相关特征,利用修饰电极检测DMSO/NaOH体系产生的超氧阴离子.试验结果表明：该修饰电极对超氧阴离子的歧化反应具有显著的催化活性,计算出异相电子传递速率常数（K_s）为0.041 cm/s,电子转移系数（α）为0.435.在0.06~0.2 μmol/L范围内,超氧阴离子浓度与峰电流呈良好的线性关系,相关系数R²为0.9719,方法检出限（LOD）为1.129×10^-3 μmol/L（S/N=3）、3.683×10^-3 μmol/L（S/N=10）,精密度试验测定得相对标准偏差（RSD,n=9）为3.83%.     

Simultaneous Stabilization of Potassium Metal and Superoxide in K–O2 Batteries on the Basis of Electrolyte Reactivity

In superoxide batteries based on O₂/O₂^? redox chemistry, identifying an electrolyte to stabilize both the alkali metal and its superoxide remains challenging owing to their reactivity towards the electrolyte components. Bis(fluorosulfonyl)imide (FSI^?) has been recognized as a “magic anion” for passivating alkali metals. The KFSI–dimethoxyethane electrolyte passivates the potassium metal anode by cleavage of S?F bonds and the formation of a KF‐rich solid–electrolyte interphase (SEI). However, the KFSI salt is chemically unstable owing to nucleophilic attack by superoxide and/or hydroxide species. On the other hand, potassium bis(trifluorosulfonyl)imide (KTFSI) is stable to KO₂, but results in mossy potassium deposits and irreversible plating and stripping. To circumvent this dilemma, we developed an artificial SEI for the metal anode and thus long‐cycle‐life K–O₂ batteries. This study will guide the development of stable electrolytes and artificial SEIs for metal–O₂ batteries.     

A Non‐Enzymatic Pathway with Superoxide in Intracellular Terpenoid Synthesis

Non‐C₅‐units terpenoids (norisoprenoids) with an acetonyl group are widely distributed in nature. However, studies on the biosynthesis of norisoprenoids are scarce. Now, the C₃₃ norisoprenoid, (all‐E)‐farnesylfarnesylacetone, was identified from Bacillus spp. and it was elucidated for the first time that superoxide mediates the cleavage of menaquinones (vitamin K) to form norisoprenoids in saponification treatment. From in vivo experiments using gene‐disrupted Bacillus subtilis strains targeted for enzymes responsible for menaquinone biosynthesis and for superoxide dismutase, it was suggested that the non‐enzymatic cleavage (autoxidation) of menaquinone with superoxide resulted in norisoprenoid synthesis in Bacillus cells. Furthermore, the bioactive norisoprenoids, farnesylacetone and phytone, were produced in Bacillus cells by this novel synthesis system.